St. Augustine grass named ‘B12’

ABSTRACT

‘B12’ is an attractive, green (RHS 137A)  Stenotaphrum secundatum  grass (St. Augustine grass), with a fast growth habit, brown with predominant yellow-green internode color, moderate internode length, and a long leaf blade that is highly infolded in mature turf, with a fine leaf appearance. ‘B12’ is also characterized by good disease and pest resistance and superior color retention under low fertilizer conditions.

Latin name of the genus and species: The Latin name of the genus andspecies of the novel variety disclosed herein is Stenotaphrumsecundatum.

Variety denomination: The inventive variety of St. Augustine grassdisclosed herein has been given the variety denomination ‘B12’.

BACKGROUND OF THE INVENTION

Stenotaphrum secundatum belongs to the grass family, Gramineae, and hasthe common names St. Augustine grass and buffalo grass. Stenotaphrumsecundatum is a vigorous-growing perennial grass that prefers warmweather climates, and is able to withstand temperatures as high as 105°F. Its market class is turf grass, and it is widely used as a lawn grassin the Southern United States where many other grasses cannot survivethe extreme temperatures.

Some commonly known varieties of S. secundatum include ‘Floratine’(unpatented), ‘Bitter Blue’ (unpatented), ‘Floratam’ (unpatented),‘Seville St. Augustine’ (unpatented), ‘Raleigh St. Augustine’(unpatented), ‘Texas Common’ (unpatented), ‘SS-100’ (U.S. Plant Pat. No.9,395; sold under the name PALMETTO™), ‘Shademaster’ (unpatented), ‘SirWalter’ (Australian PBR No. 96/226; unpatented in the United States),and ‘ST-85’ (Australian Patent No. 643567; unpatented in the UnitedStates).

-   Lineage:

The variety ‘B12’ was identified in Spring 2001 in Clarendon, New SouthWales, Australia, during a seedling selection of cultivated ‘SirWalter’. ‘Sir Walter’ is characterized by long-average internode lengthand intense purple intended color. Selection criteria for ‘B12’ weregreener internode color and shorter internode length. The parent plant‘Sir Walter’ was grown in isolation, and 5000 seeds were collected fromopen pollination in Spring 2000. These seeds were sown, and in Februaryand March 2001, twelve of the resulting plants were selected based ontheir green stems. In Spring 2001 a final single selection, designated‘B12’, was made from these twelve seedlings based on shorter internodelength.

-   Asexual Reproduction:

‘B12’ was first asexually propagated by stolons in September 2001 inClarendon, New South Wales, Australia. ‘B12’ has since been asexuallypropagated by means of stolons. The distinctive characteristics of thevariety have remained stable and true to type through successive cyclesof asexual propagation.

SUMMARY OF THE INVENTION

‘B12’ is an attractive, green (RHS 137A) Stenotaphrum secundatum grass(St. Augustine grass), with a fast growth habit, brown with predominantyellow-green internode color, moderate internode length, and a long leafblade that is highly infolded in mature turf, with a fine leafappearance. ‘B12’ is also characterized by good disease and pestresistance and superior color retention under low fertilizer conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Stolon length and color. Photo taken July 2003 in New SouthWales, Australia showing the back view of the stolon. ‘Sir Walter’ withits longer internode length, and more purple internode is on the left.‘B12’ with its medium internode length and its lighter colored internodeis in the center. ‘SS-100’ is to the right with its shorter and greenerinternode.

FIG. 2. Stenotaphrum ‘B12’ (left) with comparator ‘Sir Walter’ (center)and ‘Shademaster’ (right) showing differences in internode color.

FIG. 3. Leaf angle comparison among ‘SS-100’ (left), ‘B12’ (middle), and‘Sir Walter’ (right).

FIG. 4. ‘B12’ has a finer leaf and a higher frequency of acute leafangles. Measurements for leaf angle trial (Comparison Trial 4) came fromwithin this ring.

FIG. 5. ‘Sir Walter’ has a less fine leaf, and a higher frequency ofless acute leaf angles. Measurements for leaf angle trial (ComparisonTrial 4) came from within this ring.

FIG. 6. ‘SS100’ has a less fine leaf, and a higher frequency of moreopen leaf angles than ‘B12’. Measurements for leaf angle trial(Comparison Trial 4) came from within this ring.

FIG. 7. ‘B12’ is shown as 18-month-old turf, illustrating its fine leafappearance, with an Australian 50 cent piece in the picture.

FIG. 8. ‘Sir Walter’ is shown as 18-month-old turf, illustrating itsbroader appearing leaf as compared with ‘B12’, with an Australian 50cent piece in the picture.

FIG. 9. ‘SS100’ is shown as 18-month-old turf, illustrating its broaderappearing leaf as compared with ‘B12’, with an Australian 50 cent piecein the picture.

FIG. 10. Clippings collected from samples of each Stenotaphrum evaluatedin the low fertilizer trials. At the end of the Kincumber trial, allvegetative material was harvested from each pot, placed in a plastic bagand weighed (see Table 8). ‘B12’ and ‘SS-100’ grew better with nofertilizer as compared with the other varieties.

FIG. 11. Pots of the different Stenotaphrum showing the excellent growthof ‘B12’ and ‘SS-100’ (labeled as ‘Palmetto’) with no fertilizer. Thelong leaves of ‘B12’ can also be seen.

FIG. 12. PCR products separated on a 1.5% agarose gel inTris-borate-EDTA containing ethidium bromide. PCR amplification productswere visualized using a Bio-Rad transilluminator. Lane 1=‘Bitterblue’;Lane 2=‘SS-100’ ‘PALMETTO’™; Lane 3=‘Woerner Classic’; Lane 4=‘RaleighSt. Augustine’; Lane 5=‘Floratam’; Lane 6=‘B12’.

DETAILED BOTANICAL DESCRIPTION OF THE VARIETY

The following is a detailed botanical description of a new Stenotaphrumsecundatum grass variety known as ‘B12’, based upon observations of theplant grown in nursery pots and field plots. Those skilled in the artwill appreciate that certain characteristics will vary with older or,conversely, with younger plants. ‘B12’ has not been observed under allpossible environmental conditions. Where dimensions, sizes, colors andother characteristics are given, it is to be understood that suchcharacteristics are approximations or averages set forth as accuratelyas practicable. The phenotype of the variety may differ from thedescriptions herein with variations in the environment such as season,temperature, light intensity, day length, cultural conditions, and thelike. Color notations are based on The Royal Horticultural SocietyColour Chart, The Royal Horticultural Society, London (1995 edition).

‘B12’ is a perennial, vegetatively propagated Stenotaphrum grass,believed to be a variety of Stenotaphrum secundatum. The parent of ‘B12’is ‘Sir Walter’ (Australian PBR no. 96/226; unpatented in the UnitedStates), a variety of Stenotaphrum widely grown in Australia. ‘B12’ is afine leafed, fast-growing variety.

TECHNICAL DESCRIPTION OF THE VARIETY

The description of the variety below is taken from a pot trial conductedin the Spring/Summer 2002 in Clarendon, New South Wales, Australia.(Comparison Trial 1 below). Plants were six-months old at the time ofobservation. Additional characteristics of the plant are illustrated inFIGS. 1-11.

-   Plant characteristics: Perennial, stoloniferous grass, habit    prostrate becoming erect when flowering, culms branched, glabrous.-   Stolon: Roots at nodes, internode length (4th from tip) medium-long    (mean 48.6 mm), average internode length (internodes 4 to 6 from    tip) medium-long (mean 50.4 mm), color yellow-green (RHS 144A) at    node changing to yellow-green (RHS 148A) along internode with    diffuse brown (RHS 200A) becoming predominantly brown (RHS 200A) on    upper exposed side of internode with maturity.-   Leaf: Sheath length medium (mean 19.1 mm), color green (RHS 138B),    leaf veins parallel and obscure, blade length medium (mean 20.2 mm),    blade width medium (mean 6.4 mm), color green (RHS 137A), apex    acute, leaf margin entire (smooth).-   Ligule: Ligule is a hairy rim.-   Awns: Absent.-   Inflorescence: Spike-like panicle, length is 5.5 to 9 cm, color    green (approximately RHS 137A).-   Glumes and lemmas: Color green (RHS 137A) to yellow-green (RHS    146A); the upper glumes and lemmas are unequal in length as compared    with the lower glumes and lemmas: the lower short, blunt, nerveless;    the upper more acute and nerved.-   Anther color: Greyed orange (between RHS 167A and 167B).-   Stigma color: Purple violet (RHS 81A).-   Seed: Seed is approximately 3 mm long and is rarely produced.-   Cultural notes: ‘B12’ has been observed to survive to a temperature    of −10° Celsius, and is heat and humidity tolerant. It has good    drought tolerance, comparable with ‘Sir Walter’, and ‘SS-100’ (sold    under the name PALMETTO™; U.S. Plant Pat. No. 9,395), with a strong    and vigorous root system. ‘B12’ also grows well under low fertilizer    conditions. Shade tolerance is good and is being further evaluated.    ‘B12’ competes well with invasive weeds. It strikes well when being    stolonised, and transplants well as sod.-   Disease resistance: ‘B12’ appears to be resistant to grey-leaf spot    as the observed occurrence of this disease has been very low (see    also Table 13 below). The variety has reduced susceptibility to    fungus and heat stress, and retains good summer color (i.e., does    not show significant yellowing in summer).-   Winter color: ‘B12’ has good winter color, being one of the last    Stenotaphrum secundatum to go dormant, but under severe repeated    frost it will turn brown.

COMPARISON TRIALS WITH OTHER VARIETIES IN AUSTRALIA

A series of comparative trials were carried out among ‘B12’, ‘SirWalter’, ‘SS-100’, ‘ST-85’ (Australian Patent No. 643567; not patentedin the United States) and ‘Shademaster’ (unpatented; a commonly-grownAustralian variety).

The characteristics used to identify the most similar varieties ofcommon knowledge to ‘B12’ were: plant characteristics, main color of thestolon, internode length, and leaf width and length. Based on thesecriteria, ‘Sir Walter’ and ‘SS-100’ were selected as the most similarcomparators. For a broader comparison, although these varieties arereadily distinguishable by stolon color alone, varieties ‘ST-85’ and‘Shademaster’ were also included in some of the trials.

The results of the comparison trials are shown in Tables 1-8 below. Insummary:

-   -   ‘B12’ has a shorter internode length than ‘Sir Walter’, and a        longer internode length than ‘SS-100’ and ‘ST-85’.    -   ‘B12’ has a longer leaf than ‘ST-85’, ‘SS-100’, ‘Sir Walter’ and        ‘Shademaster’.    -   ‘B12’ has an internode color of brown with predominant yellow        green, while ‘SS100’ has a green internode, ‘Sir Walter’ has an        internode of dark purple with a little green, and ‘ST-85’ and        ‘Shademaster’ have a dark purple internode color.    -   ‘B12’ grows at a similar speed to ‘Sir Walter’, as determined by        both stolon growth across the ground and upward leaf growth.        Both grasses have similar mowing rates. ‘B12’ grows only        modestly faster across the ground from stolons than does        ‘SS100’, but grows significantly faster in the leaf, therefore        requiring more mowing than ‘SS-100’. It is noted that ‘B12’        grows unusually fast for a fine leaf Stenotaphrum secundatum.        ‘ST-85’ is a more typical fine leaf variety, which grows more        slowly than ‘B12’ in the leaf and stolon.    -   With respect to leaf width, it was determined that ‘B12’ has a        finer leaf than ‘Sir Walter’, ‘Shademaster’, and ‘SS100’.        Further, when ‘B12’ is established and forms a dense mat, it has        a more angled infolded leaf than ‘Sir Walter’ and ‘SS100’, which        gives ‘B12’ the appearance of being even more fine leafed than        these other varieties. The angled leaf of ‘B12’ enhances the        fine leafed appearance and makes it visually appear to be much        more than 1 mm finer than ‘SS-100’.    -   Under conditions of low fertilizer, the best visual appearance        ratings were for ‘B12’ and ‘SS-100’, followed by ‘Shademaster’.        The poorest visual appearance rating was for ‘Sir Walter’        followed by ‘ST-85’.    -   In general, the varieties with highest visual ratings under low        fertilizer conditions also had the highest scores for        uniformity, density and greenness.

The comparison trials and results are described in more detail below.

Australian Comparison Trial 1: Internode Color, Internode Length andLeaf Length

A comparative trial was conducted in Clarendon, New South Wales,Australia in Spring/Summer 2002. ‘B12’, ‘Sir Walter’, ‘Shademaster’,‘SS100’ and ‘ST-85’ were compared for stolon color (i.e., internodecolor), leaf length and internode length. Individual stolons were takenfrom open beds, and were then planted in 140 mm pots filled with soilesspotting mix. Nutrition was maintained with slow release fertilizers,which were added at the time the Stenotaphrum were potted. The plantswere grown in full sun in the open, with irrigation. The pots were sevenmonths old at the time of trial. The plants did not flower during thetrial. The trial design consisted of thirty pots of each varietyarranged in a completely randomized design. Measurements were taken inDecember 2002. The results are shown in Table 1 below.

TABLE 1 ‘Sir ‘Shade- ‘B12’ Walter’ master’ ‘SS100’ ‘ST-85’ InternodeBrown Dark Dark Green Dark Color with pre- purple purple Purple (overalldominant with little appearance) yellow- green green Leaf Length (mm) -4^(th) node from tip mean 20.2 17.2 15.2 14.5 10.7 standard  4.2  1.5 1.7  2 1.7 deviation LSD/sig  3.13 Ns P ≦ 0.01 P ≦ 0.01 P ≦ 0.01Average Internode Length (mm) - internodes 4 to 6 mean 50.4 57.1 52.344.1 34.85 standard  4.6  4.8  2.9  3.1 2.9 deviation LSD/sig  4.76 P ≦0.01 ns P ≦ 0.01 P ≦ 0.01

Australian Comparison Trial 2: Leaf Width and Intended Length

Another comparison trial was conducted in Clarendon, New South Wales,Australia in October 2002, wherein three one-foot square pieces of sodof ‘B12’, ‘Sir Walter’ and ‘SS-100’ were compared with respect tointernode length and leaf width. The sod was from Comparison Trial 3below, and was planted in February 2002. The ten-month old sod was grownusing standard practices in the sod industry, and was harvested with aspade. Seventy-eight random samples were taken, 26 from each piece ofsod, and were examined for internode length and leaf width. The resultsare shown in Table 2. Note that results for pot-grown samples willdiffer from turf-grown samples. For example, when the turf starts to matin the pot, its growth is constrained, which affects some measurements.In addition, mowing turf-grown samples will affect some measurements.The pot trials, however, are useful for showing trends, and the observedtrends were consistent throughout the trials.

TABLE 2 ‘B12’ ‘SIR WALTER’ ‘SS-100’ Leaf width (mm) - 4^(th) node fromtip Mean  5.7  6.633 6.3 Std deviation  1.2  1.1 1.1 LSD/sig  0.7 P ≦0.01 P ≦ 0.01 Average internode length Mean 30.04 39.87 25.23 Stddeviation 15 16.3 12.4 LSD/sig  9.1 P ≦ 0.01 ns

Australian Comparison Trial 3: Time to Harvest

In a third study, carried out in February 2002, approximately 15 squaremeters of bare ground in a plot in Clarendon, New South Wales, Australiawas planted with stolons of ‘B12’, ‘Sir Walter’, ‘SS100’ and ‘ST-85’.These plots were mown, watered and fertilized regularly, and weremonitored for 12 months. At 9 months, ‘B12’ and ‘Sir Walter’ were readyto harvest. At 9 months and 2 weeks, ‘SS100’ was ready to harvest. Both‘Sir Walter’ and ‘SS-100’ are considered fast-growing varieties ofStenotaphrum secundatum. In the same trial, ‘ST-85’ was not ready forharvest for another 2 months and 2 weeks (i.e., at 12 months). Theresults of this comparison are shown below in Table 3. Readiness forharvest was judged by ability to harvest the turf with a hand turfcutter.

TABLE 3 Variety Time from Planting to Harvest ‘B12’  9 Months ‘SirWalter’  9 Months ‘SS-100’  9 Months 2 weeks ‘ST-85’ 12 Months

Australian Comparison Trial 4: Leaf Width and Angle Comparison

In July 2003, a fourth trial comparing leaf width and leaf angle wasconducted with ‘Sir Walter’ and ‘SS-100’, the two varieties ofStenotaphrum secundatum that were determined to be the most similar to‘B12’. Upon inspection of mature, never-harvested material fromComparative Trials 2 and 3 above, it was observed that ‘B12’ had a finerleaf appearance than the leaf measurements in the first trialssuggested. For this reason, a second comparison of leaf measurements wasconducted. The results are shown in Table 4.

TABLE 4 ‘B12’ ‘SIR WALTER’ ‘SS-100’ Leaf width (mm) - 4^(th) node fromtip Mean 4.85 6.02 5.88 Std deviation 0.8 0.9 0.7 LSD/sig 0.48 P ≦ 0.01P ≦ 0.01

As expected, the comparison demonstrated that the leaf of ‘B12’ wasfiner than the leaves of the other two varieties. Nonetheless, it wassurprising that the measurements indicated that ‘B12’ was only about 1mm finer in leaf blade width. Based on visual appraisal of thevarieties, ‘B12’ would appear to be at least 2 mm to 2.5 mm finer. Uponcloser inspection and evaluation, it was noted that the leaf of ‘B12’was far more infolded than the leaf of ‘Sir Walter’ and ‘SS100’. Asystem of measuring was devised by bending small pieces of wire at 40degrees, 80 degrees, 120 degrees, and 150 degrees. A series ofmeasurements was taken from each grass. All samples had leaf angles thatfell below 180 degrees. Any sample that could not be clearly measured(e.g., borderline cases) was discarded. In total, six samples werediscarded: three from ‘SS-100’, two from ‘Sir Walter’ and one from‘B12’.

The results of this study can be seen in Table 5 below. It was foundthat ‘B12’ was more tightly infolded than the other varieties, as moreof its leaves were folded at shaper angles, and less were folded at openangles. The somewhat finer leaf of ‘B12’, in combination with the moreacute infold angle of the leaf, results in ‘B12’ having a much finerappearance than ‘Sir Walter’ and ‘SS-100’.

TABLE 5 0 to 40° 41 to 80° 121 to 151 to Extremely Highly 81 to 120°150° 180° Angle angled angled Angled Open Very open Category 5 4 3 2 1Frequency 6 16 13 4 1 of angle for ‘B12’ Frequency 1 8 12 12 7 of anglefor ‘Sir Walter’ Frequency 3 10 16 8 3 of angle for ‘SS-100’

Australian Comparison Trials under Low Fertilizer Conditions

Stenotaphrum varieties held at Kincumber, New South Wales, Australiasince December 2002 were rated for performance on Mar. 21, 2003. Theturf was in 140 mm pots and was transferred to Kincumber from the plantsused in Comparison Trial 1, described above. The turf had not receivedfertilizer and was minimally watered. The plants were cut back (to potedge) approximately four weeks prior to evaluation. Each variety wasassessed for the indices shown in Table 6.

TABLE 6 No. Visual Uni- flower- Leaf Green- Variety Reps rating formitying length Density ness ‘B12’ 4 8-9  8-9 25% long  8-9 light green (RHS144A) ‘Shade- 4 7-8  8-9  0% med- 8 light master’ long green (RHS 144A)‘SS- 4 8-9 9  0% med- 9 medium 100’ long green (RHS 146A) ‘Sir 4 5-6 525% med-  5-6 yellow- Walter’ long green (RHS 144A- B) ‘ST 85’ 2 5-6 8100%  short 8 light green (RHS 144B) Performance Ratings: Visual rating:0 = dead, 10 = vigorous habit Uniformity: 0 = poor, 10 = perfectly evenover all replicates Density: 0 = very sparse, 10 = very dense Greenness:yellow-green (chlorotic, nutrient deficient), light green, medium green,dark green (no nutrition deficiencies apparent).

The remaining trial stock at Clarendon, New South Wales, Australia fromComparison Trial 1 was evaluated in the same way on Mar. 26, 2003. Theseplants were adequately watered, but were not fertilized or pruned.Results are shown in Table 7.

TABLE 7 No. Visual Uni- flower- Leaf Green- Variety Reps rating formitying length Density ness ‘B12’ 20 9 9 100% long 8-9 light green (RHS144A) ‘Shade- 20 8 8  25% med 8  light master’ green (RHS 144A) ‘SS- 209 9 100% med- 8  medium 100’ long green (RHS 146A) ‘Sir 20  5-6  5-6 50% med- 5-6 yellow- Walter’ long green (RHS 144A- B) ‘ST-85’ 20  6-7 9100% short 8-9 Light green (RHS 144B) Performance Ratings: Visualrating: 0 = dead, 10 = vigorous habit Uniformity: 0 = poor, 10 =perfectly even over all replicates Density: 0 = very sparse, 10 = verydense Greenness: yellow green (chlorotic, nutrient deficient), lightgreen, medium green, dark green (no nutrition deficiencies apparent).

The Kincumber stock (from the study shown in Table 6) was then harvestedto determine average shoot yield as assessed by measuring the freshweight of all above ground parts (leaves and stolons). Results are shownin Table 8.

TABLE 8 Mean Shoot Variety Fresh Weight (g) ‘B12’ 80 ‘Shademaster’ 71‘SS-100’ 105 ‘Sir Walter’ 39 ‘ST 85’ 55

Conclusions of Low Fertilizer Trials

The best visual appearance ratings were for ‘B12’ and ‘SS-100’, followedby ‘Shademaster’. The poorest visual appearance rating was for ‘SirWalter’ followed by ‘ST-85’.

In general, the varieties with highest visual ratings also had thehighest scores for uniformity, density and greenness. In other words,these varieties have complete ground coverage, even growth and a greenerappearance than the poorer varieties.

Since the plants in these studies were not mown, it appears that longerleaf length combined with uniform growth generally contribute to apositive visual assessment. Leaf length correlated with shoot mass forthe longer leaf varieties ‘B12’ and ‘SS-100’.

Comparison Trials with other Varieties in the Southern United States

In April 2004, comparative evaluations began for the purpose of furtherquantifying characteristics of ‘B12’. Twenty-two evaluation sites acrossthe southern United States were selected to host these evaluations(sites were located in Virginia, North Carolina, South Carolina,Georgia, Florida, Alabama, Louisiana, Arkansas, Texas, New Mexico andArizona). Each site was constructed to a specified protocol thatcomprised: pre-plant herbicide application, plot dimensioning andconfiguration, replication instruction, establishment regimen, andmaintenance guidelines.

At each site, ‘B12’ was compared to other local “standard” St. Augustinegrass varieties (each site used two or three standard varieties forcomparison, specifically ‘SS-100’ (sold under the name ‘PALMETTO’™),‘Raleigh St. Augustine’ and/or ‘Floratam’). All sites were constructedwith three replications of each variety used. Plots measured 8′×8′ andobservations were made on 30 day intervals, beginning 30 days afterplanting (DAP). Evaluation criteria included Overall Turf Quality,Color, Presence of Seed Head/Flowers, Ground Coverage, Internode Length,Disease Presence, and Insect Damage.

-   90 DAP Results.-    In August 2004, observations from eight representative sites (see    Table 9) were collected and analyzed. When compared to observations    made on the standard St. Augustine grass varieties, several    distinguishing characteristics of ‘B12’ became apparent. These    findings are discussed below:    -   Overall Turf Quality    -    Compared to St. Augustine grass varieties ‘SS-100’, ‘Raleigh        St. Augustine’ and ‘Floratam’, ‘B12’ was characterized as        developing a higher overall turf quality at the 30, 60 and 90        DAP observations (see Table 10).    -   Color    -    ‘B12’ consistently exhibited a more pleasing genetic color than        ‘SS-100’, ‘Raleigh St. Augustine’ or ‘Floratam’. This        characteristic was most notable during 60 and 90 DAP        observations (see Table 11).    -   Ground Coverage    -    The rate at which ‘B12’ grew to cover its test plots was        significantly faster than that of ‘SS-100’ or ‘Raleigh St.        Augustine’. Although the difference was not as significant when        compared to ‘Floratam’, an increased coverage rate was also        observed (see Table 12).    -   Disease    -    The occurrence of disease, specifically gray-leaf spot, was        notably lower in ‘B12’ and ‘Raleigh St. Augustine’ at 90 DAP. At        60 DAP; ‘B12’, ‘SS-100’ and ‘Raleigh St. Augustine’ all had a        much lower occurrence of gray-leaf spot than ‘Floratam’ (see        Table 13).    -   Internode Length    -    ‘B12’ has a longer internode length than ‘SS-100’, ‘Raleigh St.        Augustine’ or ‘Floratam’ when measured between nodes 3 and 4        from the tip of the stolon (see Table 14).

TABLE 9 Comparative Evaluation Sites Site Site Varieties Evaluated #Location ‘B12’ ‘SS-100’ ‘Raleigh’ ‘Floratam’ 1 Kenansville, FL Yes YesNo Yes 2 Arcadia, FL Yes Yes No Yes 3 Awendaw, SC Yes Yes Yes No 4Raeford, NC Yes Yes Yes No 5 Boling, TX Yes Yes Yes No 6 Pilot Point, TXYes Yes Yes No 7 San Antonio, TX Yes Yes Yes No 8 Crosby, TX Yes Yes YesNo

TABLE 10 Overall Turf Quality (Average on 1-9 Scale; 9 = Ideal Turf)Days After St. Augustine Planting (DAP) Grass Variety 30 60 90 ‘B12’ 7.47.8 7.9 ‘SS-100’ 6.8 6.8 6.2 ‘Raleigh’ 7.1 7.1 6.8 ‘Floratam’ 6.5 5.35.2

TABLE 11 Color (Average on 1-9 Scale; 9 = Ideal Color) Days After St.Augustine Planting (DAP) Grass Variety 30 60 90 ‘B12’ 7.3 7.5 7.6‘SS-100’ 6.8 6.8 6.4 ‘Raleigh’ 7.2 7.1 6.9 ‘Floratam’ 6.7 5.2 6.3

TABLE 12 Ground Coverage (Average Percent Cover) Days After St.Augustine Planting (DAP) Grass Variety 30 60 90 ‘B12’ 31% 75% 93%‘SS-100’ 21% 45% 66% ‘Raleigh’ 24% 47% 69% ‘Floratam’ 27% 68% 90%

TABLE 13 Disease (Average Disease Presence on 1-9 Scale; 9 = No Disease)Days After St. Augustine Planting (DAP) Grass Variety 30 60 90 ‘B12’ 9.07.6 7.7 ‘SS-100’ 9.0 7.9 6.8 ‘Raleigh’ 9.0 7.9 7.7 ‘Floratam’ 9.0 5.35.2

TABLE 14 Internode Length (Average from 3^(rd) to 4^(th) in millimeters)Days After St. Augustine Planting (DAP) Grass Variety 60 90 ‘B12’ 50.858.4 ‘SS-100’ 38.1 48.3 ‘Raleigh’ 48.3 45.7 ‘Floratam’ 43.2 48.3

Summary

Comparative observations of ‘B12’ were taken as part of an on-goingmulti-site, geographically diverse, evaluation regimen which began inApril 2004. Analysis of these 30, 60, and 90 DAP observations clearlyidentified characteristics of ‘B12’ that differentiate it from ‘SS-100’,‘Raleigh’ and ‘Floratam’ St. Augustine grasses. Notably, enhanced TurfQuality and Color augments the marketability of ‘B12’ as producers andconsumers generally prefer turfgrass varieties that are moreaesthetically pleasing. Increased Ground Coverage rates reduceproduction and overhead costs and provide turfgrass producers withlarger profit margins and quicker harvest cycles. Also, reducedoccurrence of Disease, specifically gray-leaf spot, lessens the need forfungicide inputs during production and end-use; reducing costs andminimizing potential environmental impacts from chemical applications.Finally, a distinguishing physical characteristic of ‘B12’ is its longerinternode Length (as measured between the 3^(rd) and 4^(th) internode).

Comparative DNA Analysis of ‘B12’ with other Turfgrasses

Randomly Amplified Polymorphic DNA (RAPD) analysis of ‘B12’ incomparison with other turfgrasses was performed using a series often-mer primers from Operon Technologies, Inc. (Alameda, Calif.) asdescribed below:

-   Plant material

Samples from six different turfgrasses were provided by Todd Bunnell,(Clemson University). The turfgrass varieties used in the analysis were:‘Bitterblue’, ‘PALMETTO’™, ‘Woerner Classic’, ‘Raleigh St. Augustine’,‘Floratam’, and ‘B12’.

-   DNA isolation

DNA was isolated from the leaf blades using the DNeasy procedure fromQiagen (Valencia, Calif.). The DNA extracts were quantified so thatequal amounts of DNA could be used in the amplifications.

-   Amplification primers

One hundred ten-mer primers from Operon Technologies, Inc. (Alameda,Calif.) were used in the comparisons among the six turf grass samples.Of these, 32 were used to evaluate the complete set of samples (OPB 1,OPB 6, OPB 7, OPB 11, OPB 12, OPB 15, OPB 17, OPB 18, OPB 19, OPC 4, OPC6, OPC 8, OPC 9, OPC 10, OPC 11, OPC 12, OPJ 7, OPJ 9, OPJ 10, OPJ 11,OPJ 13, OPK 1, OPK 4, OPK 10, OPK 11, OPK 15, OPAC 2, OPAC 3, OPAC 10,OPAC 11, OPAC 18, OPAC 19, OPAC 20)

FIG. 12 shows the results when the primer OPC4 (CCGCATCTAC; SEQ ID NO:1)was used.

-   Amplification

The PCR reaction was carried out using TaKaRa Taq polymerase (Takara BioInc.) in the manufacturer's supplied buffer with a final concentrationof MgCl₂ of 2.5 mM. Each reaction contained 25 μg of leaf DNA. The PCRreactions were subjected to a hot start with the buffer heated to 85° C.before the addition of Taq polymerase. All the reactions were carriedout using MJR PTC-100 thermal cyclers (MJ Research, Inc.).

The amplification program consisted of:

-   -   1. 1 min 96° C.    -   2. 1 min 94° C.    -   3. 1 min 35° C.    -   4. 1 min 72° C.    -   5. Cycle to step 2 45 times.    -   6. Hold at 72° C. for 10 minutes.    -   7. Cool to 4° C.    -   8. Hold.

-   Gel electrophoresis and photography

The PCR products were separated on a 1.5% agarose gel inTris-borate-EDTA containing ethidium bromide and visualized using aBio-Rad transilluminator. Images were captured with a Kodak DC290camera. Lane 1=‘Bitterblue’; Lane 2=‘PALMETTO’™; Lane 3=‘WoernerClassic’; Lane 4=‘Raleigh St. Augustine’; Lane 5=‘Floratam’; Lane6=‘B12’.

-   RAPD analysis

Randomly Amplified Polymorphic DNA (RAPD) analysis of the six turfgrasssamples was carried out as described above. All of the samples could bedistinguished from each other using one or more of the ten-mer primers.The results with the OPC4 primer are shown in FIG. 12. As shown in thefigure, ‘B12’ can be distinguished by RAPD analysis from the otherturfgrasses evaluated using the OPC4 primer.

1. A new and distinct variety of Stenotaphrum secundatum plant named‘B12’, substantially as illustrated and described herein.